背景 肿瘤细胞对传统化学治疗药物产生耐药性是视网膜母细胞瘤(RB)化学疗法失败的主要原因,如何提高化学治疗药物的敏感性是RB治疗过程中亟待解决的问题,已有研究表明全反式维甲酸(ATRA)能够抑制肿瘤细胞的生长,但其是否能够提高肿瘤细胞对传统化学治疗药物的敏感性尚不清楚.目的 观察ATRA联合长春新碱对SO-RB50细胞增生的抑制作用. 方法 对SO-RB50细胞进行常规培养,培养液中分别添加不同浓度的ATRA或不同质量浓度的长春新碱,用细胞计数试剂盒-8(CCK-8)法分别测定ATRA及长春新碱作用48 h后对SO-RB50细胞的半数抑制浓度(IC50).将常规培养的SO-RB50细胞分为正常对照组、ATRA组、长春新碱组和联合用药组,分别将不同药物根据IC50量添加至培养液进行细胞培养,采用CCK-8法于培养后每24小时检测细胞增生情况,连续测定6d,绘制细胞生长曲线.用流式细胞仪检测各组细胞用药72h后不同细胞周期的比例,annexin V/碘化丙啶(PI)法检测各组药物处理48 h后SO-RB50细胞的凋亡情况. 结果 ATRA作用于SO-RB50细胞的IC50值为12.84 μmol/L,长春新碱为0.11 μg/ml.正常对照组SO-RB50细胞生长曲线随着培养时间的延长逐渐上升,但ATRA和长春新碱处理后细胞增生曲线平缓,以联合用药组细胞增生量最低,曲线最平缓.药物作用48 h,正常对照组、ATRA组、长春新碱组和联合用药组细胞的吸光度(A450)分别为1.078±0.022、0.611 ±0.038、0.596±0.031和0.483±0.030,药物作用72 h分别为1.380±0.021、0.799±0.016、0.668±0.041和0.532±0.033,总体比较差异有统计学意义(F分组=1 115.207,P=0.000;F时间=257.781,P=0.000),ATRA组、长春新碱组和联合用药组A450值均明显低于正常对照组,差异均有统计学意义(均P=0.000).SO-RB50细胞经药物处理后72 h,各组细胞周期发生变化,与正常对照组比较,ATRA组G0/G1期细胞比例显著增加,S期细胞百分数显著减少,长春新碱组G0/G1期细胞百分数减少,而G2/M期细胞比例显著增加,总体比较差异有统计学意义(FG0/G1=130.565、Fs=57.435、FG2/M=114.290,均P＜0.05).SO-RB50细胞经药物处理48 h后,正常对照组、ATRA组、长春新碱组及联合用药组细胞的凋亡率分别为(7.17±0.18)％、(27.34±1.36)％、(27.49±2.45)％和(34.50± 1.84)％,总体差异有统计学意义(F=147.555,P＜0.05),联合用药组的细胞凋亡率显著高于正常对照组、ATRA组和长春新碱组,差异均有统计学意义(均P=0.000).结论 ATRA可以在一定程度上提高SO-RB50细胞对长春新碱治疗的敏感性,2种药物的联合应用可增强对SO-RB50细胞的抑制作用,其作用机制可能与参与细胞周期调控、促进细胞凋亡有关.
揹景 腫瘤細胞對傳統化學治療藥物產生耐藥性是視網膜母細胞瘤(RB)化學療法失敗的主要原因,如何提高化學治療藥物的敏感性是RB治療過程中亟待解決的問題,已有研究錶明全反式維甲痠(ATRA)能夠抑製腫瘤細胞的生長,但其是否能夠提高腫瘤細胞對傳統化學治療藥物的敏感性尚不清楚.目的 觀察ATRA聯閤長春新堿對SO-RB50細胞增生的抑製作用. 方法 對SO-RB50細胞進行常規培養,培養液中分彆添加不同濃度的ATRA或不同質量濃度的長春新堿,用細胞計數試劑盒-8(CCK-8)法分彆測定ATRA及長春新堿作用48 h後對SO-RB50細胞的半數抑製濃度(IC50).將常規培養的SO-RB50細胞分為正常對照組、ATRA組、長春新堿組和聯閤用藥組,分彆將不同藥物根據IC50量添加至培養液進行細胞培養,採用CCK-8法于培養後每24小時檢測細胞增生情況,連續測定6d,繪製細胞生長麯線.用流式細胞儀檢測各組細胞用藥72h後不同細胞週期的比例,annexin V/碘化丙啶(PI)法檢測各組藥物處理48 h後SO-RB50細胞的凋亡情況. 結果 ATRA作用于SO-RB50細胞的IC50值為12.84 μmol/L,長春新堿為0.11 μg/ml.正常對照組SO-RB50細胞生長麯線隨著培養時間的延長逐漸上升,但ATRA和長春新堿處理後細胞增生麯線平緩,以聯閤用藥組細胞增生量最低,麯線最平緩.藥物作用48 h,正常對照組、ATRA組、長春新堿組和聯閤用藥組細胞的吸光度(A450)分彆為1.078±0.022、0.611 ±0.038、0.596±0.031和0.483±0.030,藥物作用72 h分彆為1.380±0.021、0.799±0.016、0.668±0.041和0.532±0.033,總體比較差異有統計學意義(F分組=1 115.207,P=0.000;F時間=257.781,P=0.000),ATRA組、長春新堿組和聯閤用藥組A450值均明顯低于正常對照組,差異均有統計學意義(均P=0.000).SO-RB50細胞經藥物處理後72 h,各組細胞週期髮生變化,與正常對照組比較,ATRA組G0/G1期細胞比例顯著增加,S期細胞百分數顯著減少,長春新堿組G0/G1期細胞百分數減少,而G2/M期細胞比例顯著增加,總體比較差異有統計學意義(FG0/G1=130.565、Fs=57.435、FG2/M=114.290,均P＜0.05).SO-RB50細胞經藥物處理48 h後,正常對照組、ATRA組、長春新堿組及聯閤用藥組細胞的凋亡率分彆為(7.17±0.18)％、(27.34±1.36)％、(27.49±2.45)％和(34.50± 1.84)％,總體差異有統計學意義(F=147.555,P＜0.05),聯閤用藥組的細胞凋亡率顯著高于正常對照組、ATRA組和長春新堿組,差異均有統計學意義(均P=0.000).結論 ATRA可以在一定程度上提高SO-RB50細胞對長春新堿治療的敏感性,2種藥物的聯閤應用可增彊對SO-RB50細胞的抑製作用,其作用機製可能與參與細胞週期調控、促進細胞凋亡有關.
배경 종류세포대전통화학치료약물산생내약성시시망막모세포류(RB)화학요법실패적주요원인,여하제고화학치료약물적민감성시RB치료과정중극대해결적문제,이유연구표명전반식유갑산(ATRA)능구억제종류세포적생장,단기시부능구제고종류세포대전통화학치료약물적민감성상불청초.목적 관찰ATRA연합장춘신감대SO-RB50세포증생적억제작용. 방법 대SO-RB50세포진행상규배양,배양액중분별첨가불동농도적ATRA혹불동질량농도적장춘신감,용세포계수시제합-8(CCK-8)법분별측정ATRA급장춘신감작용48 h후대SO-RB50세포적반수억제농도(IC50).장상규배양적SO-RB50세포분위정상대조조、ATRA조、장춘신감조화연합용약조,분별장불동약물근거IC50량첨가지배양액진행세포배양,채용CCK-8법우배양후매24소시검측세포증생정황,련속측정6d,회제세포생장곡선.용류식세포의검측각조세포용약72h후불동세포주기적비례,annexin V/전화병정(PI)법검측각조약물처리48 h후SO-RB50세포적조망정황. 결과 ATRA작용우SO-RB50세포적IC50치위12.84 μmol/L,장춘신감위0.11 μg/ml.정상대조조SO-RB50세포생장곡선수착배양시간적연장축점상승,단ATRA화장춘신감처리후세포증생곡선평완,이연합용약조세포증생량최저,곡선최평완.약물작용48 h,정상대조조、ATRA조、장춘신감조화연합용약조세포적흡광도(A450)분별위1.078±0.022、0.611 ±0.038、0.596±0.031화0.483±0.030,약물작용72 h분별위1.380±0.021、0.799±0.016、0.668±0.041화0.532±0.033,총체비교차이유통계학의의(F분조=1 115.207,P=0.000;F시간=257.781,P=0.000),ATRA조、장춘신감조화연합용약조A450치균명현저우정상대조조,차이균유통계학의의(균P=0.000).SO-RB50세포경약물처리후72 h,각조세포주기발생변화,여정상대조조비교,ATRA조G0/G1기세포비례현저증가,S기세포백분수현저감소,장춘신감조G0/G1기세포백분수감소,이G2/M기세포비례현저증가,총체비교차이유통계학의의(FG0/G1=130.565、Fs=57.435、FG2/M=114.290,균P＜0.05).SO-RB50세포경약물처리48 h후,정상대조조、ATRA조、장춘신감조급연합용약조세포적조망솔분별위(7.17±0.18)％、(27.34±1.36)％、(27.49±2.45)％화(34.50± 1.84)％,총체차이유통계학의의(F=147.555,P＜0.05),연합용약조적세포조망솔현저고우정상대조조、ATRA조화장춘신감조,차이균유통계학의의(균P=0.000).결론 ATRA가이재일정정도상제고SO-RB50세포대장춘신감치료적민감성,2충약물적연합응용가증강대SO-RB50세포적억제작용,기작용궤제가능여삼여세포주기조공、촉진세포조망유관.
Background Drug resistance is the main cause of failure after chemotherapy of retinoblastoma (RB),and how to improve the sensitivity of RB cells to chemotherapic drug become an urgent issue.All trans retinoic acid (ATRA) can inhibit the growth of tumor cells.However,whether ATRA increases the sensitivity of RB cells to chemotherapic drug is unclear.Objective This study aimed to investigate the inhibitory effect of ATRA with vincristine on the proliferation of SO-RB50 cells.Methods SO-RB50 cells were cultured routinely.Different concentrations of ATRA or vincristine were added into the medium for 48 hours for the determination of IC50 by cell counting kit-8 (CCK-8) method.Cultured cells were divided into normal control group,ATRA group,vincristine group and combined drug group.The cells were treated by ATRA or vincristine with the dose of IC50,and the proliferation of the cells was detected every day at the 24-hour interval for 6 consecutive days.The percentage of the cells in different cell cycles was analyzed 72 hours after treatment using flow cytometry.Cell apoptosis rate was detected and calculated 48 hours after treatment by annexin V/propidium iodide (PI) method.Results The IC50 of ATRA was approximately 12.84 μ mol/L,and that of vincristine was 0.11 μg/ml.The growth curve of SO-RB50 cells was gradually raised as the lapse of time,but the curves were relatively low in the ATRA group,vincristine group and combined drug group,with the lowest curve in the combined drug group.The proliferation values of the cells (A450)were 1.078±0.022,0.611 ±0.038,0.596 ±0.031 and 0.483 ±0.030 in 48 hours after treatment,and those in 72 hours were 1.380± 0.021,0.799 ± 0.016,0.668 ± 0.041 and 0.532 ± 0.033 in normal control group,ATRA group,vincristine group and combined drug group,showing significant differences among the groups and various time points (Fgroup =1115.207,P =0.000;Ftime =257.781,P =0.000).The A450 values of the ATRA group,vincristine group and combined drug group were significantly lower than those of normal control group (all at P＜0.05).The percentage of the cells in different cell cycles was changed after 72 hours' treatment and the differences were statistically significant (FG0/G1 =130.565,Fs =57.435;FG2/M =114.290,P＜0.05).Compared with the normal control group,the percentage of G0/G1 phase cells was increased and S phase cells was decreased significantly in ATRA group,the percentage of G0/G1 phase cells was decreased and G2/M phase cells was increased significantly in vincristine group(P＜0.05).The apoptotic rate of the cells was (7.17±0.18) ％,(27.34±1.36) ％,(27.49±2.45) ％ and (34.50±1.84) ％ in normal control group,ATRA group,vincristine group and combined drug group,with a significant difference among the groups (F=147.555,P＜0.05),and the apoptotic rate in the combined drug group was remarkedly lower than that of normal control group,ATRA group and vincristine group (all at P=0.000).Conclusions ATRA can improve the sensitivity of SO-RB50 cells to chemotherapeutic drug.The combined application of ATRA and vincristine enhance the inhibitory effect on the cells probably by regulating cell cycle and inducing apoptosis.
